Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-11-20
2002-11-12
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007200, C435S007900, C435S007930, C436S538000, C436S541000
Reexamination Certificate
active
06479246
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a competitive binding assay kit and process for the immunological detection of a presence of a blood antigen of specified phenotype (for example phenotypes of HPA) in a sample of whole blood.
BACKGROUND OF THE INVENTION
Human Platelet Antigen 1 (HPA1), also known as PLA1, is a polymorphic determinant on the glycoprotein (GP) IIb/IIIa complex, that resides at the N-terminal region of GPIIIa. The great majority of the Caucasian population is homozygous or heterozygous for HPA1a (about 72% and 26% respectively) (1). However, 2-3% of the population is homozygous for HPA1b (PLA1 negative) putting them at risk from developing antibodies to HPA1a either during pregnancy or following transfusion with blood containing HPA1a platelets. For example, HPA1b pregnant women carrying HPA1a positive babies may produce anti-HPA1a antibodies which could lead to fetal or neonatal alloimmune thrombocytopenia (FAITP or NAITP) respectively, with 50% of the cases occurring at first pregnancy. One outcome of FAITP/NAITP, which has a frequency of 1 in 1000-2000 births, is intracranial hemorrhage with 6.5-10% mortality and 20% morbidity (neurological sequelae) (2).
Moreover, HPA1b individuals (mostly elderly multi-parous women) transfused with blood containing HPA1a platelets may develop post-transfusion purpura (PTP) which is characterized with severe thrombocytopenia, 5-10 days following the transfusion. Although not as common as NAITP (only about 200 cases reported world-wide), PTP is nevertheless associated with 10-20% morbidity/mortality (3).
The treatment and management of FAITP/NAITP and PTP relies basically on several infusions of intra-venous IgG with or without transfusion with HPA1b platelets. In view of the serious or fatal outcomes of FAITP/NAITP and PTP and their increasingly effective treatment, antenatal screening for HPA1b is becoming widely accepted as an important prophylactic measure (4,5). For such wide-scale screening, established assays such as monoclonal antibody immobilization of platelet antigens (MAIPA), platelet suspension immunofluorescence test, or flow cytometry are not suitable due to their technical or financial burdens (6-8). Recently, Metcalfe et al reported a simplified method for large-scale HPA1a phenotyping for antenatal screening which was adapted from MAIPA (9). However, the Metcalfe et al assay relies on the extraction of glycoprotein GPIIb/IIIa antibody complex from platelets pre-incubated with the antibody. The assay is also considerably time consuming requiring a number of incubation and centrifugation steps.
EP 0028133 describes a method of detecting and quantitating haptens and antigens. A competitve binding assay is described in which a sample and a reagent containing an immunoreactive antibody are in a liquid phase, the sample and reagent either being mixed together or reacted before contacting a solid phase onto which a known concentration of a substance selected from haptens, antigens and/or mixtures thereof, which are also reactive with said antibody, is bound.
There is thus a requirement for an immunoassay which does not require a sample of whole blood to be substantially purified.
It is an object of the present invention to obviate and/or mitigate some of the above disadvantages.
Generally speaking the present invention provides a simple and potentially automatable competitive binding assay and assay kit for determining the presence in vitro of a specified blood antigen (for instance HPA1a) in a sample of whole blood. It is generally based on the surprising feature that the assay is able to determine a presence or absence of a specified blood antigen in a sample of whole blood. It might have been expected that cells and other species normally present in whole blood would interfere with such an assay.
SUMMARY OF THE INVENTION
The present invention provides in a first aspect a method of performing a competitive binding immunoassay for determining in vitro the presence of a specified blood antigen phenotype in a sample of whole blood, which comprises:
mixing the sample of whole blood with a predetermined amount of antibody specific to said blood antigen phenotype, so as to bind the antibody to any of said antigen present in the sample of whole blood;
bringing the mixture into contact with a substrate upon which antigen of said phenotype has been immobilized such as to bind any remaining unbound antibody to said immobilized antigen, thereby forming an immobilized antigen-antibody complex;
washing the immobilized antigen-antibody complex and;
estimating the amount of immobilized complex and consequently the specified blood antigen phenotype.
The present invention in a further aspect provides a competitive binding immunoassay kit for determining the presence of a specified blood antigen phenotype in a sample of whole blood which, comprises;
a substrate having immobilized thereon substantially pure blood antigen of said phenotype.
a supply of antibody for specifically binding to said antigen, and
means for enabling detection of said specifically bound antibody.
The immunoassay of the present invention is described as a competitive binding immunoassay essentially because any blood antigen of said specified phenotype present in the whole blood sample competes with said immobilized antigen, for the predetermined amount of antibody.
The immunoassay can be used to detect the presence of any suitable intrinsic blood cell antigen of a specified phenotype (excluding foreign species such as viral antigens etc.). The blood antigens include antigenic determinants on platelet glycoproteins, in particular HPA1 antigen of
1
a
or
1
b
phenotype. Other antigens which are also suitable are glycoproteins or other determinants on blood cells, particularly white blood cells (such as granulocytes).
The sample of whole blood may be obtained fresh from an individual to be tested, or the blood may be from whole blood stored up to 25 days at 4° C. (for example citrated or EDTA whole blood). The whole blood sample may be used untreated, or alternatively the whole blood sample can be subjected to a washing step prior to testing (e.g. by diluting the blood, centrifuging it and resuspending in a buffer).
Typically, a sample of serum containing a high titer of antibody specific to said blood antigen may be employed in the immunoassay. Alternatively a monoclonal antibody specific to said blood antigen could be used.
The substrate upon which said substantially pure antigen of said phenotype is immobilized, can be any suitable substrate known in the art. Typically this can be paper, plastics such as nitrocellulose, or glass (e.g. microscope slides). Most preferably the substrate is a well of a plastics microtiter plate, as commonly employed in immunoassays.
The antigen to be immobilized is preferably substantially pure. That is, the antigen is purified away from other blood antigens or blood components that could affect the immunoassay. It is important that the antigen be substantially pure if the antibody used is in a serum sample. This is to prevent any additional antibodies that may be present in the serum sample, from binding to their respective blood antigen. If a monoclonal antibody is employed, it may not be necessary to highly purify the antigen, depending on the degree of cross-reactivity of the monoclonal antibody which should be specific to its specified antigen. The antigen used should be purified from a blood source, different to that being tested. This is to minimize any further antigen-antibody complexes which may be formed, from interfering with the immunoassay.
Typically a number of immune (i.e. antigen-antibody) complexes may be formed when bringing the mixture into contact with the substrate, for instance; a) specified antibody and immobilized antigen, b) specified antibody and antigen present in the whole blood, and c) non-specifically bound unspecified antibody to immobilized antigen. The washing serves to remove all complexes except the specified antibody and immobilized antigen complex.
The mixing of the sam
Bessos Hagop
Murphy William Gerrard
Common Services Agency
Myers Bigel & Sibley & Sajovec
Stucker Jeffrey
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